Identification of New Hematopoietic Cell Subsets with a Polyclonal Antibody Library Specific for Neglected Proteins

The identification of new markers, the expression of which defines new phenotipically and functionally distinct cell subsets, is a main objective in cell biology. We have addressed the issue of identifying new cell specific markers with a reverse proteomic approach whereby approximately 1700 human open reading frames encoding proteins predicted to be transmembrane or secreted have been selected in silico for being poorly known, cloned and expressed in bacteria. These proteins have been purified and used to immunize mice with the aim of obtaining polyclonal antisera mostly specific for linear epitopes. Such a library, made of about 1600 different polyclonal antisera, has been obtained and screened by flow cytometry on cord blood derived CD34+CD45dim cells and on peripheral blood derived mature lymphocytes (PBLs). We identified three new proteins expressed by fractions of CD34+CD45dim cells and eight new proteins expressed by fractions of PBLs. Remarkably, we identified proteins the presence of which had not been demonstrated previously by transcriptomic analysis. From the functional point of view, looking at new proteins expressed on CD34+CD45dim cells, we identified one cell surface protein (MOSC-1) the expression of which on a minority of CD34+ progenitors marks those CD34+CD45dim cells that will go toward monocyte/granulocyte differentiation. In conclusion, we show a new way of looking at the membranome by assessing expression of generally neglected proteins with a library of polyclonal antisera, and in so doing we have identified new potential subsets of hematopoietic progenitors and of mature PBLs

Schematic representation of the antisera library generation.

<p>Schematic representation of the antisera library generation.</p

FACS analysis with sera specific for well-known proteins.

<p>(A) Comparison of the CD8 staining performed on PBL with either a commercially available anti CD8 mAb (BD biosciences) or the anti CD8 alpha antiserum at 1∶100 dilution points. Both the samples were stained also with commercially available anti CD3 and anti CD4 mAb (BD biosciences). The distribution of CD4 and CD8 is analyzed upon gating on CD3 positive cells. (B) Examples of staining with antisera from the library. PBLs from healthy donors were stained with anti CD2, CD1d, CD8 alpha, CD25, CD72, CD80, CD38, and CD86. The expression of CD25 was assessed upon a 24 hours activation of PBLs with 1 µg/ml of PHA. The expression of CD80 and CD86 was assessed upon gating on monocytes after a 24 hours activation of PBLs with 1 µg/ml of PHA. The expression of CD133 was analyzed on cord blood derived CD34+, CD45dim cells. Serum from not immunized mice was used as negative control in all the stainings.</p

Results of sera screening by FACS on PBL and Cord Blood cells.

<p>(A) FACS analysis of sera positive on PBLs. PBLs were stained with the indicated sera at the optimal dilution point (1∶50 to 1∶200). The samples were stained also with anti CD3, anti CD19 and anti CD56 mAbs to analyze the sera reactivity upon gating on the different subpopulations. A plot representative of five different donors is shown for each serum. (B) KRTCAP-3 specific serum recognizes PHA-treated cells. PBMCs are treated for 24 hours with 1 µg/ml of PHA. After the treatment both un-stimulated and treated cells are stained with the KRTCAP-3-specific serum. (C) FACS analysis of sera positive on cord blood cells. Cord blood mononuclear cells are stained with the indicated sera at the optimal concentration (1∶50 to 1∶100). The samples are stained also with anti CD45 and anti CD34 mAbs to perform the analysis upon gating on CD34highCD45dim. A plot representative of a least 3 independent donors is shown. Il all the cases (A,B,C,) a staining with the serum of not immunized mice was used as negative control. (D) RT-PCR analysis. a- cDNA from total PBMC were amplified with primers specific for the indicated proteins. b- cDNA from both un-stimulated and PHA-treated PBMC was amplified with KRTCAP-3 specific primers. KRTCAP3 expression is up regulated two to three times. Beta actin amplification is used as normalization. c- cDNA samples from CD34+CD45dim cells were generated by retro-transcription of RNA extracted from a pool of CD34 positive cells from 2–3 independent cord blood units magnetically purified using the Miltenyi CD34 microbeads kit according the manufacturer instruction. The purity of the CD34+CD45dim cells was usually >99%. The samples were amplified with primers specific for the indicated proteins and described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034395#s2" target="_blank">Methods</a> section.</p

Assessment of antisera specificity on Hela transfected cells.

<p>Hela cells were transiently transfected with a myc-tag version of the proteins identified with the sera library. At 24 hours from the transfection cells were lysated as described in the Method section. 40 µg of total proteins were loaded on SDS page and a WB analysis was performed using both an anti myc mAb (9E10 clone) and the corresponding antiserum. (A) WB analysis of Hela cells transfected with CRISP-1 and MOSC-1. In both the cases the anti myc mAb and the specific antiserum recognized a protein of the expected molecular weight that is not present in the cells transfected with the mock vector. A comparable result was obtained wit KRTCAP-3 (B), TMCC-1 (C), TMEM38B (D) and SUSD3 (E) transfected cells. The WB analysis of GSG1-L cells (E) and LPPR2 cells (F) shows that neither the anti myc nor the specific antiserum is able to recognize in a specific way a protein in transfected cells.</p

Comparison of the results obtained with antisera specific for well-known proteins assessed both by FACS on PBMCs from healthy donors and by IHC on inflamed lymph nodes.

<p>Comparison of the results obtained with antisera specific for well-known proteins assessed both by FACS on PBMCs from healthy donors and by IHC on inflamed lymph nodes.</p

Genome-wide analysis of DNA methylation, copy number variation, and gene expression in monozygotic twins discordant for primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an uncommon autoimmune disease with a homogeneous clinical phenotype that reflects incomplete disease concordance in monozygotic (MZ) twins. We have taken advantage of a unique collection consisting of genomic DNA and mRNA from peripheral blood cells of female MZ twins (n = 3 sets) and sisters of similar age (n = 8 pairs) discordant for disease. We performed a genome-wide study to investigate differences in (i) DNA methylation (using a custom tiled four-plex array containing tiled 50-mers 19,084 randomly chosen methylation sites), (ii) copy number variation (CNV) (with a chip including markers derived from the 1000 Genomes Project, all three HapMap phases, and recently published studies), and/or (iii) gene expression (by whole-genome expression arrays). Based on the results obtained from these three approaches we utilized quantitative PCR to compare the expression of candidate genes. Importantly, our data support consistent differences in discordant twins and siblings for the (i) methylation profiles of 60 gene regions, (ii) CNV of 10 genes, and (iii) the expression of 2 interferon-dependent genes. Quantitative PCR analysis showed that 17 of these genes are differentially expressed in discordant sibling pairs. In conclusion, we report that MZ twins and sisters discordant for PBC manifest particular epigenetic differences and highlight the value of the epigenetic study of twins

Mildly compromised tetrahydrobiopterin cofactor biosynthesis due to Pts variants leads to unusual body fat distribution and abdominal obesity in mice

Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, alkylglycerol monooxygenase, and nitric oxide synthases (NOS). Inborn errors of BH4 metabolism lead to severe insufficiency of brain monoamine neurotransmitters while augmentation of BH4 by supplementation or stimulation of its biosynthesis is thought to ameliorate endothelial NOS (eNOS) dysfunction, to protect from (cardio-) vascular disease and/or prevent obesity and development of the metabolic syndrome. We have previously reported that homozygous knock-out mice for the 6-pyruvolytetrahydropterin synthase (PTPS; Pts-ko/ko) mice with no BH4 biosynthesis die after birth. Here we generated a Pts-knock-in (Pts-ki) allele expressing the murine PTPS-p.Arg15Cys with low residual activity (15% of wild-type in vitro) and investigated homozygous (Pts-ki/ki) and compound heterozygous (Pts-ki/ko) mutants. All mice showed normal viability and depending on the severity of the Pts alleles exhibited up to 90% reduction of PTPS activity concomitant with neopterin elevation and mild reduction of total biopterin while blood L-phenylalanine and brain monoamine neurotransmitters were unaffected. Yet, adult mutant mice with compromised PTPS activity (i.e., Pts-ki/ko, Pts-ki/ki or Pts-ko/wt) had increased body weight and elevated intra-abdominal fat. Comprehensive phenotyping of Pts-ki/ki mice revealed alterations in energy metabolism with proportionally higher fat content but lower lean mass, and increased blood glucose and cholesterol. Transcriptome analysis indicated changes in glucose and lipid metabolism. Furthermore, differentially expressed genes associated with obesity, weight loss, hepatic steatosis, and insulin sensitivity were consistent with the observed phenotypic alterations. We conclude that reduced PTPS activity concomitant with mildly compromised BH4-biosynthesis leads to abnormal body fat distribution and abdominal obesity at least in mice. This study associates a novel single gene mutation with monogenic forms of obesity